Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

Bioinspired fabrication of biosilica-based bone substitution materials

Until today, autogenic bone grafts from various donor regions represent the gold standard in the field of bone reconstruction, providing both osteoinductive and osteoconductive characteristics. However, due to low availability and a disequilibrium between supply and demand, the risk of disease transfer and morbidity, usually associated with autogeneic bone grafts, the development of biomimic materials with structural and chemical properties similar to those of natural bone have been extensively studied. So far,rnonly a few synthetic materials, so far, have met these criteria, displaying properties that allow an optimal bone reconstitution. Biosilica is formed enzymatically under physiological-relevant conditions (temperature and pH) via silicatein (silica protein), an enzyme that was isolated from siliceous sponges, cloned, and prepared in a recombinant way, retaining its catalytic activity. It is biocompatible, has some unique mechanical characteristics, and comprises significant osteoinductive activity.rnTo explore the application of biosilica in the fields of regenerative medicine,rnsilicatein was encapsulated, together with its substrate sodium metasilicate, into poly(D,L-lactide)/polyvinylpyrrolidone(PVP)-based microspheres, using w/o/wrnmethodology with solvent casting and termed Poly(D,L-lactide)-silicatein silicacontaining-microspheres [PLASSM]. Both silicatein encapsulation efficiency (40%) and catalytic activity retention upon polymer encapsulation were enhanced by addition of an essential pre-emulsifying step using PVP. Furthermore, the metabolic stability, cytoxicity as well as the kinetics of silicatein release from the PLASSM were studied under biomimetic conditions, using simulated body fluid. As a solid support for PLASSM, a polyvinylpyrrolidone/starch/Na2HPO4-based matrix (termed plastic-like filler matrix containing silicic acid [PMSA]) was developed and its chemical and physical properties determined. Moreover, due to the non-toxicity and bioinactivity of the PMSA, it is suggested that PMSA acts as osteoconductive material. Both components, PLASSM and PMSA, when added together, form arnbifunctional 2-component implant material, that is (i)non-toxic(biocompatible), (ii)moldable, (iii) self-hardening at a controlled and clinically suitable rate to allows a tight insertion into any bone defect (iv) biodegradable, (v)forms a porous material upon exposure to body biomimetic conditions, and (vi)displays both osteoinductive (silicatein)and osteoconductive (PMSA) properties.rnPreliminary in vivo experiments were carried out with rabbit femurs, by creatingrnartificial bone defects that were subsequently treated with the bifunctional 2-component implant material. After 9 weeks of implantation, both computed tomography (CT) and morphological analyses showed complete resorption of the implanted material, concurrent with complete bone regeneration. The given data can be considered as a significant contribution to the successful introduction of biosilica-based implants into the field of bone substitution surgery.

Alpha, Omega end group functionalization of RAFT polymers based on pentafluorophenyl esters and methane thiosulfonates

While polymers with different functional groups along the backbone have intensively been investigated, there is still a challenge in orthogonal functionalization of the end groups. Such well-defined systems are interesting for the preparation of multiblock (co) polymers or polymer networks, for bio-conjugation or as model systems for examining the end group separation of isolated polymer chains. rnHere, Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization was employed as method to investigate improved techniques for an a, w end group functionalization. RAFT produces polymers terminated in an R group and a dithioester-Z group, where R and Z stem from a suitable chain transfer agent (CTA). rnFor alpha end group functionalization, a CTA with an activated pentafluorophenyl (PFP) ester R group was designed and used for the polymerization of various methacrylate monomers, N-isopropylacrylamide and styrene yielding polymers with a PFP ester as a end group. This allowed the introduction of inert propyl amides, of light responsive diazo compounds, of the dyes NBD, Texas Red, or Oregon Green, of the hormone thyroxin and allowed the formation of multiblocks or peptide conjugates. rnFor w end group functionalization, problems of other techniques were overcome through an aminolysis of the dithioester in the presence of a functional methane thiosulfonate (MTS), yielding functional disulfides. These disulfides were stable under ambient conditions and could be cleaved on demand. Using MTS chemistry, terminal methyl disulfides (enabling self-assembly on planar gold surfaces and ligand substitution on gold and semiconductor nanoparticles), butynyl disulfide end groups (allowing the “clicking” of the polymers onto azide functionalized surfaces and the selective removal through reduction), the bio-target biotin, and the fluorescent dye Texas Red were introduced into polymers. rnThe alpha PFP amidation could be performed under mild conditions, without substantial loss of DTE. This way, a step-wise synthesis produced polymers with two functional end groups in very high yields. rnAs examples, polymers with an anchor group for both gold nanoparticles (AuNP) and CdSe / ZnS semi-conductor nanoparticles (QD) and with a fluorescent dye end group were synthesized. They allowed a NP decoration and enabled an energy transfer from QD to dye or from dye to AuNP. Water-soluble polymers were prepared with two different bio-target end groups, each capable of selectively recognizing and binding a certain protein. The immobilization of protein-polymer-protein layers on planar gold surfaces was monitored by surface plasmon resonance.Introducing two different fluorescent dye end groups enabled an energy transfer between the end groups of isolated polymer chains and created the possibility to monitor the behavior of single polymer chains during a chain collapse. rnThe versatility of the synthetic technique is very promising for applications beyond this work.